Stabilization of dehydrogenases with stable coenzymes

ABSTRACT

The present invention relates to a method for stabilizing an enzyme by storing the enzyme in the presence of a stable coenzyme. The present invention further relates to an enzyme stabilized with a stable coenzyme, and to the use thereof in test elements for detecting analytes.

CLAIM OF PRIORITY

The present application is a continuation application based on andclaiming priority to PCT/EP2009/001206, filed Feb. 19, 2009, whichclaims priority to European Application No. 08003054.7, filed Feb. 19,2008, each of which are hereby incorporated by reference in theirrespective entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for stabilizing an enzyme bystoring the enzyme in the presence of a stable coenzyme. The presentinvention further relates to an enzyme stabilized with a stablecoenzyme, and to the use thereof in test elements for detectinganalytes.

BACKGROUND

Biochemical measurement systems are important constituents of clinicallyrelevant methods of analysis. The priority here is to measure analytes,e.g. metabolites or substrates, which are determined directly orindirectly with the aid of an enzyme. The analytes are in this caseconverted with the aid of an enzyme-coenzyme complex and thenquantified. This entails the analyte to be determined being brought intocontact with a suitable enzyme and a coenzyme, with the enzyme usuallybeing employed in catalytic amounts. The coenzyme is changed, e.g.oxidized or reduced, by the enzymatic reaction. This process can bedetected directly, or electrochemically or photometrically through amediator. A calibration provides a direct relationship between themeasurement and the concentration of the analyte to be determined.

Coenzymes are organic molecules which are bound covalently ornon-covalently to an enzyme and which are changed by the conversion ofthe analyte. Prominent examples of coenzymes are nicotinamide adeninedinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate(NADP), respectively, from which NADH and NADPH, respectively, areproduced by reduction.

Measurement systems known in the prior art are notable for being stablefor a limited period and for the specific requirements on theenvironment, such as cooling or dry storage, for achieving thisstability. For particular applications, e.g. tests carried out by thefinal user himself, such as, for example, in the self-monitoring ofblood glucose, it is therefore possible for incorrect results to occurthrough incorrect, unnoticed faulty storage. The exhaustion ofdesiccants through the primary packaging being opened for too long inparticular may lead to faulty measurements which, with some systems, canscarcely be identified by the user.

One known measure employed to increase the stability of biochemicalmeasurement systems is the use of stable enzymes, e.g. the use ofenzymes from thermophilic organisms. A further possibility is tostabilize enzymes by chemical modification, e.g. crosslinking, or bymutagenesis. In addition, enzyme stabilizers such as, for example,trehalose, polyvinylpyrrolidone and serum albumin can also be added, orthe enzymes can be enclosed e.g. by photopolymerization in polymernetworks.

Attempts have also been made to improve the stability of biochemicalmeasurement systems by using stable mediators. Thus, the specificity oftests is increased, and interference during the reaction is eliminated,through the use of mediators with a redox potential which is as low aspossible. However, the redox potentials of the enzyme/coenzyme complexesform a lower limit for the redox potential of mediators. Below thesepotentials, the reaction with the mediators is slowed down or evenstopped.

An alternative possibility is also to use biochemical measurementsystems without mediators, in which for example there is directdetection of coenzymes, e.g. of the coenzyme NADH. One disadvantage ofsuch measurement systems is, however, that coenzymes such as NAD andNADP are unstable.

NAD and NADP are base-labile molecules whose degradation pathways aredescribed in the literature (N. J. Oppenheimer in The PyridineNucleotide Coenzymes, Academic Press New York, London 1982, editors J.Everese, B. Anderson, K. You, Chapter 3, pages 56-65). The degradationof NAD and NADP, respectively, essentially results in ADP-ribose throughcleavage of the glycosyl linkages between the ribose and the pyridineunit. The reduced forms NADH and NADPH are on the other handacid-labile: e.g. epimerization is one known degradation pathway. Inboth cases, the instability of NAD/NADP and NADH/NADPH derives from thelability of the glycosyl linkage between the ribose unit and thepyridine unit. However, even under conditions which are not drastic,such as, for example, in aqueous solution, the coenzymes NAD and NADP,respectively, are hydrolysed solely through the ambient moisture. Thisinstability may lead to inaccuracies in the measurement of analytes.

A number of NAD/NADP derivatives is described for example in B. M.Anderson in The Pyridine Nucleotide Coenzymes, Academic Press New York,London 1982, editors J. Everese, B. Anderson, K. You, Chapter 4. Most ofthese derivatives are, however, not well accepted by enzymes. The onlyderivative which has to date therefore been used for diagnostic tests is3 acetylpyridine adenine dinucleotide (acetyl NAD) which was describedfor the first time in 1956 (N. O. Kaplan, J. Biol. Chem. (1956), 221,823). Also this coenzyme shows a poor acceptance by enzymes and a changein the redox potential.

WO 01/94370 describes the use of further NAD derivatives with a modifiedpyridine group. Modifications of the nicotinamide group have, however,in general a direct influence on the catalytic reaction. In most cases,this influence is negative.

In a further idea for stabilization, the ribose unit has been altered inorder thereby to influence the stability of the glycosyl linkage. Thisprocedure does not directly interfere with the catalytic reaction of thenicotinamide group. However, there may be an indirect influence as soonas the enzyme exhibits a strong and specific binding to the ribose unit.Kaufmann et al. disclose in this connection in WO 98/33936 and U.S. Pat.No. 5,801,006, and in WO 01/49247, respectively, a number ofthioribose-NAD derivatives. A connection between the modification of thenicotinamide-ribose unit and the activity of the derivatives inenzymatic reactions has, however, not been shown to date.

CarbaNAD, a derivative without a glycosyl linkage, was described for thefirst time in 1988 (J. T. Slama, Biochemistry 1989, 27, 183 andBiochemistry 1989, 28, 7688). The ribose therein is replaced by acarbocyclic sugar unit. Although carbaNAD was described as a substrateof dehydrogenases, its activity has not to date been demonstratedclinically in biochemical detection methods.

A similar approach was described later by G. M. Blackburn, Chem. Comm.,1996, 2765, in order to prepare carbaNAD with a methylenebisphosphonatecompound instead of the natural pyrophosphate. Themethylenebisphosphonate shows increased stability towards phosphatasesand was used as inhibitor of ADP-ribosyl cyclase. An increase inhydrolysis stability was not the aim (J. T. Slama, G. M. Blackburn).

WO 2007/012494 and U.S. Ser. No. 11/460,366 disclose stable NAD/NADH andNADP/NADPH derivatives, respectively, enzyme complexes of thesederivatives and the use thereof in biochemical detection methods andreagent kits.

An object of the present invention is to provide methods for stabilizingenzymes, especially for the long-term stabilization of enzymes.

SUMMARY

This object and others that will be appreciated by those of ordinaryskill in the art are achieved by a method for stabilizing an enzyme,where the enzyme is stored in the presence of a stable coenzyme. It hassurprisingly been found that long-term stabilization of several weeks ormonths at high relative moisture or even in liquid phase and at elevatedtemperatures is possible with the aid of a stable coenzyme. Thisperception is surprising because it is known that although enzymes havean increased short-term stability for some hours in the presence ofnative coenzyme (Bertoldi et al., Biochem. J. 389, (2005), 885-898; vanden Heuvel et al., (J. Biol. Chem. 280 (2005), 32115-32121; and Pan etal., (J. Chin. Biochem. Soc. Vol. 3 (1974), pp. 1-8), they have shown alower stability over a longer period (Nutrition Reviews 36 (1978),251-254).

Compared with these perceptions in relation to the prior art, it wassurprising that an enzyme has a distinctly increased long-term stabilityin the presence of a stable coenzyme than does an enzyme in the presenceof a native coenzyme, especially since the stable coenzymes have a lowerbinding constant with the enzyme than does the native coenzyme.

The enzyme stabilized by the method of the invention is acoenzyme-dependent enzyme. Examples of suitable enzymes aredehydrogenases selected from a glucose dehydrogenase (E.C.1.1.1.47),lactate dehydrogenase (E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase(E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6), alcoholdehydrogenase (E.C.1.1.1.1), alpha-hydroxybutyrate dehydrogenase,sorbitol dehydrogenase or amino-acid dehydrogenase, e.g. L-amino-aciddehydrogenase (E.C.1.4.1.5). Further suitable enzymes are oxidases suchas, for instance, glucose oxidase (E.C.1.1.3.4) or cholesterol oxidase(E.C.1.1.3.6) and amino transferases, respectively, such as, forexample, aspartate or alanine amino transferase, 5′-nucleotidase orcreatine kinase. The enzyme in certain embodiments is glucosedehydrogenase.

In certain other embodiments of the invention, a mutated glucosedehydrogenase is employed. The term “mutant” as used in the context ofthe present application refers to a genetically modified variant of anative enzyme which, while the number of amino acids is the same, has anamino acid sequence which is modified compared with the wild-typeenzyme, i.e. differs in at least one amino acid from the wild-typeenzyme. The introduction of the mutation(s) can take placesite-specifically or non-site-specifically, preferably site-specificallyby using recombinant methods known in the art, whereas, appropriate forthe particular requirements and conditions, at least one amino acidexchange within the amino acid sequence of the native enzyme results.The mutant in yet other embodiments has an increased thermal orhydrolytic stability compared with the wild-type enzyme.

The mutated glucose dehydrogenase can in principle comprise the aminoacid(s) which is (are) modified by comparison with the correspondingwild-type glucose dehydrogenase at any position in its amino acidsequence. The mutated glucose dehydrogenase in certain embodimentsincludes a mutation at least one of positions 96, 170 and 252 of theamino acid sequence of the wild-type glucose dehydrogenase, withexemplary embodiments comprising mutants with mutations at position 96and position 170, and mutations at position 170 and position 252. It hasproved advantageous for these particular embodiments for the mutatedglucose dehydrogenase to comprise no further mutations besides thesemutations.

The mutation at positions 96, 170 and 252 can in principle include anyamino acid exchange which leads to a stabilization, e.g. an increase inthe thermal or hydrolytic stability, of the wild-type enzyme. Themutation at position 96 may include includes an amino acid exchange ofglutamic acid for glycine, whereas in relation to position 170 an aminoacid exchange of glutamic acid for arginine or lysine, in particular anamino acid exchange of glutamic acid for lysine, is possible. Inrelation to the mutation at position 252, this typically includes anamino acid exchange of lysine for leucine.

The mutated glycose dehydrogenase can be obtained by mutation of awild-type glucose dehydrogenase derived from any biological source,where the term “biological source” includes in the context of thisinvention both prokaryotes such as, for example, bacteria, andeukaryotes such as, for example, mammals and other animals. Thewild-type glucose dehydrogenase may be derived from a bacterium, such asglucose dehydrogenase from Bacillus megaterium, Bacillus subtilis orBacillus thuringiensis.

In one embodiment of the present invention, the mutated glucosedehydrogenase is a glucose dehydrogenase obtained by mutation ofwild-type glucose dehydrogenase from Bacillus subtilis, which has theamino acid sequence depicted in SEQ ID No.: 1 (GlucDH_E96G_E170K) orthat depicted in SEQ ID No.: 2 (GlucDH_E170K_K252L).

The stable coenzyme is a coenzyme which has been chemically modified bycomparison with the native coenzyme and which has a higher stabilitythan the native coenzyme (e.g. hydrolytic stability). The stablecoenzyme is generally stable to hydrolysis under test conditions.Compared with the native coenzyme, the stable coenzyme may have areduced binding constant for the enzyme, for example a binding constantreduced by a factor of 2 or more.

Examples of stable coenzymes are stable derivatives of nicotinamideadenine dinucleotide (NAD/NADH) or nicotinamide adenine dinucleotidephosphate (NADP/NADPH), or truncated NAD derivatives, e.g. without theAMP moiety or with non-nucleoside residues, e.g. hydrophobic residues.Likewise another example of stable coenzyme in the context of thepresent invention is the compound of the formula (I)

Exemplary stable derivatives of NAD/NADH and NADP/NADPH are described inthe aforementioned references, the disclosures of each of which arehereby expressly incorporated by reference herein in their respectiveentireties. Particular stabilized coenzymes are described in WO2007/012494 and U.S. Ser. No. 11/460,366, respectively, the disclosuresof which are hereby expressly incorporated by reference herein in theirentireties. The stable coenzyme in one embodiment is selected fromcompounds having the general formula (II)

with

A=adenine or an analogue thereof,

T=in each case independently O, S,

U=in each case independently OH, SH, BH₃ ⁻, BCNH₂ ⁻,

V=in each case independently OH or a phosphate group, or two groupsforming a cyclic phosphate group;

W═COOR, CON(R)₂, COR, CSN(R)₂ with R=in each case independently H orC₁-C₂-alkyl,

X¹, X²=in each case independently O, CH₂, CHCH₃, C(CH₃)₂, NH, NCH₃,

Y═NH, S, O, CH₂,

Z=a linear or cyclic organic radical, with the proviso that Z and thepyridine residue are not linked by a glycosidic linkage, or a salt or,where appropriate, a reduced form thereof.

In one embodiment, Z in the compounds of the formula (II) comprises alinear radical having 4-6 C atoms, for example 4 C atoms, in which 1 or2 C atoms are optionally replaced by one or more heteroatoms selectedfrom O, S and N. In other embodiments, Z comprises a radical including acyclic group which has 5 or 6 C atoms and which optionally comprises aheteroatom selected from O, S and N and optionally one or moresubstituents, and a radical CR⁴ ₂, where CR⁴ ₂ is bonded to the cyclicgroup and to X², with R⁴=in each case independently H, F, Cl, CH₃.

In yet other embodiments, Z comprises a saturated or unsaturatedcarbocyclic or heterocyclic 5-membered ring, in particular a compound ofthe general formula (III)

where a single or double bond may be present between R^(5′) and R^(5″),with

R⁴=in each case independently H, F, Cl, CH₃,

R⁵═CR⁴ ₂,

where R^(5′)═O, S, NH, NC₁-C₂-alkyl, CR⁴ ₂, CHOH, CHOCH₃, and

R^(5″)═CR⁴ ₂, CHOH, CHOCH₃ if there is a single bond between R^(5′) andR^(5″), and

where R^(5′)═R^(5″)═CR⁴ if there is a double bond between R^(5′) andR^(5″), and

R⁶, R^(6′)=in each case independently CH or CCH₃.

In one embodiment, the compounds of the invention comprise adenine oradenine analogues such as, for example, C₈- and N₆-substituted adenine,deaza variants such as 7-deaza, aza variants such as 8-aza orcombinations such as 7-deaza or 8-aza or carbocyclic analogues such asformycin, whereas the 7-deaza variants may be substituted in position 7by halogen, C₁-C₆-alkynyl, -alkenyl or -alkyl.

In a further embodiment, the compounds comprise adenosine analogueswhich, instead of ribose, comprise for example 2-methoxydeoxyribose,2′-fluorodeoxyribose, hexitol, altritol and polycyclic analogues,respectively, such as bicyclo-, LNA- and tricyclo-sugars.

It is possible in particular in the compounds of the formula (II) alsofor (di)phosphate oxygens to be replaced isotronically, such as, forexample, O⁻ by S⁻ and BH₃ ⁻, respectively, O by NH, NCH₃ and CH₂,respectively, and ═O by ═S.

In one embodiment, W in the compounds of the formula (II) of theinvention comprises CONH₂ or COCH₃.

In another embodiment, R⁵ in the groups of the formula (III) comprisesCH₂. In yet a further embodiment, R^(5′) is selected from CH₂, CHOH andNH. In yet other embodiments, R^(5′) and R^(5″) each comprise CHOH. Inyet a further embodiment, R^(5′) is NH and R^(5″) is CH₂. Specificexamples of stabilized coenzymes are depicted in FIGS. 1A and B.

In a typical embodiment, the stable coenzyme is carbaNAD.

The method of the invention is generally suitable for long-termstabilization of enzymes. This means that the enzyme stabilized with astable coenzyme shows when stored, e.g. as dry substance, for exampleover a period of at least 2 weeks, but generally at least 4 weeks and inmany cases at least 8 weeks. Furthermore, such stabilization generallyresults in a decline in enzyme activity of less than 50%, typically lessthan 30% and in many cases less than 20% in relation to the initialenzyme activity.

The method of the invention further includes a storage of the enzymestabilized with a stable coenzyme at elevated temperatures, for exampleat a temperature of at least 20° C., typically at least 25° C., and inmany cases at least 30° C. The enzyme activity in this case declines byless than 50%, typically less than 30% and in many cases less than 20%in relation to its initial level.

It is possible by the stabilization according to the invention to storethe enzyme stabilized with a stable coenzyme even without a dryingreagent for a long time, as indicated above, and/or at hightemperatures, as indicated above. It is further possible for thestabilized enzyme also to be stored at a high relative humidity, e.g. arelative humidity of at least 50%, in which case the enzyme activitydeclines by less than 50%, typically less than 30% and in many casesless than 20% in relation to the initial level.

The storage of the enzyme stabilized with a stable coenzyme can takeplace on the one hand as dry substance and on the other hand in liquidphase. The storage of the stabilized enzyme in one embodiment takesplace on or in a test element suitable for determining an analyte. Theenzyme stabilized with a stable coenzyme is in this case typically aconstituent of a detection reagent which may where appropriate alsocomprise further constituents such as, for example, salts, buffers, etc.In some embodiments, the detection reagent is generally free of amediator.

The enzyme stabilized with a stable coenzyme can be employed fordetecting analytes, for example parameters in body fluids such as, forinstance, blood, serum, plasma or urine, and in sewage samples or foodproducts, respectively.

Analytes which can be determined are any biological or chemicalsubstances which can be detected by a redox reaction, e.g. substanceswhich are substrates of a coenzyme-dependent enzyme or are themselvescoenzyme-dependent enzymes. Examples of analytes are glucose, lacticacid, malic acid, glycerol, alcohol, cholesterol, triglycerides,ascorbic acid, cysteine, glutathione, peptides, urea, ammonium,salicylate, pyruvate, 5′-nucleotidase, creatine kinase (CK), lactatedehydrogenase (LDH), carbon dioxide etc. The analyte in one particularembodiment is glucose.

Another embodiment of the present invention relates to the use of acompound of the invention or of an enzyme stabilized with a stablecoenzyme according to the invention for detecting an analyte in a sampleby an enzymatic reaction. The detection of glucose with the aid ofglucose dehydrogenase (GlucDH) is a typical example in this regard.

The alteration in the stable coenzyme by reaction with the analyte canin principle be detected in any way. It is possible in principle toemploy here all methods known from prior art for detecting enzymaticreactions. However, the alteration in the coenzyme in one embodiment isdetected by optical methods. Optical detection methods include forexample the measurement of absorption, fluorescence, circular dichroism(CD), optical rotatory dispersion (ORD), refractometry etc.

An optical detection method which may be used in the context of thepresent application is photometry. Photometric measurement of analteration in the coenzyme as a result of reaction with the analyterequires, however, the additional presence of at least one mediatorwhich increases the reactivity of the reduced coenzyme and makes itpossible for electrons to be transferred to a suitable optical indicatoror an optical indicator system.

Mediators suitable for the purposes of the present invention are interalia nitrosoanilines such as, for example,[(4-nitrosophenyl)imino]dimethanol hydrochloride, quinones such as, forexample, phenanthrenequinones, phenanthrolinequinones orbenzo[h]quinolinequinones, phenazines such as, for example,1-(3-carboxypropoxy)-5-ethylphenazinium trifluoromethanesulfonate,or/and diaphorase (EC 1.6.99.2). Preferred examples ofphenanthrolinequinones include 1,10-phenanthroline-5,6-quinones,1,7-phenanthroline-5,6-quinones, 4,7-phenanthroline-5,6-quinones, andthe N-alkylated and N,N′-dialkylated salts thereof, with preference ascounter ion in the case of N-alkylated and N,N′-dialkylated salts,respectively, for halides, trifluoromethanesulfonate or other anionswhich increase the solubility.

It is possible to use as optical indicator or as optical indicatorsystem any substance which is reducible and on reduction experiences adetectable change in its optical properties such as, for example,colour, fluorescence, reflectance, transmission, polarization or/andrefractive index. Determination of the presence or/and the amount of theanalyte in the sample can take place with the unaided eye or/and bymeans of a detection device using a photometric method which appears tobe suitable to a person skilled in the art.

Heteropolyacids and in particular 2,18-phosphomolybdic acid areoptionally used as optical indicators and are reduced to thecorresponding heteropoly blue.

The alteration in the coenzyme in other embodiments is detected bymeasuring the fluorescence. Fluorescence measurement is highly sensitiveand makes it possible to detect even low concentrations of the analytein miniaturized systems.

An alternative possibility is also to detect the alteration in thecoenzyme electrochemically using a suitable test element such as, forexample, an electrochemical test strip. The precondition for this isonce again the use of suitable mediators which can be converted by thereduced coenzyme, by transfer of electrons, into a reduced form. Theanalyte is determined by measuring the current which is needed toreoxidize the reduced mediator and which correlates with theconcentration of the analyte in the sample. Examples of mediators whichcan be used for electrochemical measurements include in particular theaforementioned mediators employed for photometric measurements.

It is possible to use a liquid test to detect an analyte, in which casethe reagent is for example in the form of a solution or suspension in anaqueous or nonaqueous liquid or as powder or lyophilisate. However, itis also possible to use a dry test, in which case the reagent is appliedto a support, a test strip. The support may include for example a teststrip including an absorbent or/and swellable material which is wettedby the sample liquid to be investigated.

In one particular embodiment, a test format includes the use of theenzyme glucose dehydrogenase with a stable NAD derivative for detectingglucose, in which case a derivative of the reduced coenzyme NADH isformed. NADH is detected by optical methods, e.g. by photometric orfluorometric determination after UV excitation. An exemplary test systemis described in US 2005/0214891, the disclosure of which is incorporatedherein by reference in its entirety.

The present invention further relates also to an enzyme stabilized witha stable coenzyme, where the stabilized enzyme shows a decline in theenzymatic activity of less than 50%, typically less than 30% and in manycases less than 20% compared with the initial level, upon storage for atleast 2 weeks, typically at least 4 weeks and in many cases at least 8weeks at a temperature of at least 20° C., typically at least 25° C. andin many cases at least 30° C., where appropriate with high humidity, andalso in certain embodiments so stored without a drying reagent.

The invention further relates also to a detection reagent fordetermining an analyte which comprises an enzyme stabilized with astable coenzyme as indicated above. The invention additionally relatesto a test element which comprises an enzyme stabilized according to theinvention and a detection reagent, respectively, according to theinvention. The detection reagent and the test element, respectively, maybe suitable for carrying out dry or liquid tests. The test element inone embodiment comprises a test strip for fluorometric or photometricdetection of an analyte. Such a test strip comprises the enzymestabilized with a stable coenzyme and immobilized on an absorbent or/andswellable material such as, for instance, cellulose, plastics, etc.

The invention is to be explained in more detail by the following figuresand examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1A is a depiction of the stable coenzyme carba-NAD (cNAD).

FIG. 1B is a depiction of the stable coenzyme pyrrolidinyl-NAD.

FIGS. 2A-2D are depictions of the results of the glucose dehydrogenaseenzyme kinetics in the presence of NAD and in the presence of cNAD,respectively, before and after storage.

FIG. 2A depicts kinetics of GlucDH in the presence of NAD after 1 day.

FIG. 2B depicts kinetics of GlucDH in the presence of cNAD after 1 day.

FIG. 2C depicts kinetics of GlucDH in the presence of NAD after storageat 32° C. and 85% relative humidity for 5 weeks.

FIG. 2D depicts kinetics of GlucDH in the presence of cNAD after storageat 32° C. and 85% relative humidity for 5 weeks.

FIG. 3 is a comparison of the blank values for glucose dehydrogenase inthe presence of NAD and of GlucDH in the presence of cNAD, respectively,over a period of up to 5 weeks at 32° C. and 85% humidity.

FIG. 4 is a depiction of various function plots of glucose dehydrogenaseafter storage of glucose dehydrogenase in the presence of NAD at 32° C.and 85% humidity. The storage time varied between 1 day and 5 weeks.

FIG. 5 is a depiction of various function plots of glucose dehydrogenaseafter storage of glucose dehydrogenase in the presence of cNAD at 32° C.and 85% humidity. The storage time varied between 1 day and 5 weeks(FIG. 5A) and between 1 day and 24 weeks (FIG. 5B), respectively.

FIG. 6 is a depiction of the residual content of NAD and cNAD afterstorage of glucose dehydrogenase in the presence of NAD and cNAD,respectively, at 32° C. and 85% humidity for 24 weeks.

FIG. 7 is a depiction of the GlucDH activity after storage of glucosedehydrogenase in the presence of NAD and cNAD, respectively, at 32° C.and 85% humidity for 5 weeks (FIG. 7A) and 24 weeks (FIG. 7B),respectively.

FIG. 8 is a depiction of the GlucDH activity after storage of glucosedehydrogenase (GlucDH wt), the double mutant GlucDH_E96G_E170K (GlucDHMut1) and the double mutant GlucDH_E170K_K252L (GlucDH Mut2) in thepresence of NAD and cNAD, respectively, at 32° C. and 83% relativehumidity over a period of 25 weeks.

FIG. 9 shows an analysis of glucose dehydrogenase by gel electrophoresisafter storage in the presence of NAD and cNAD, respectively. Testconditions: MW, 10 220 kDa markers; 1: GlucDH/NAD, 5 weeks at 6° C.; 2:GlucDH/NAD, 5 weeks at 32° C./85% relative humidity; 3: GlucDH/cNAD, 5weeks at 6° C.; 4: GlucDH/cNAD, 5 weeks at 32° C./85% relative humidity.

FIG. 10 shows an analysis of glucose dehydrogenase by gelelectrophoresis after storage at 50° C. in the presence of NAD and cNAD,respectively. Test conditions: MW, 10 220 kDa markers; 1: GlucDH 8.5mg/ml, NAD, 0 hours; 2: GlucDH 8.5 mg/ml, NAD, 22 hours; 3: GlucDH 8.5mg/ml, NAD, 96 hours; 4: GlucDH 8.5 mg/ml, NAD, 118 hours; 5: GlucDH 8.5mg/ml, NAD, 140 hours; 6: GlucDH 8.5 mg/ml, NAD, 188 hours; 7: GlucDH8.5 mg/ml, NAD, 476 hours; 8: GlucDH 8.5 mg/ml, cNAD, 0 hours; 9: GlucDH8.5 mg/ml, cNAD, 188 hours; 10: GlucDH 8.5 mg/ml, cNAD, 476 hours.

FIG. 11 is a depiction of the stability of glucose dehydrogenase in thepresence of NAD and cNAD, respectively, in liquid phase at 50° C. over aperiod of 4 days (FIG. 11A) and 14 days (FIG. 11B), respectively. Testconditions: GlucDH 10 mg/ml; NAD and cNAD, respectively, 12 mg/ml;buffer: 0.1 M Tris, 1.2 M NaCl, pH 8.5; temperature 50° C.

FIG. 12 shows an analysis of alcohol dehydrogenase from yeast by gelelectrophoresis after storage in the presence of NAD and cNAD,respectively. Test conditions: MW, 10 220 kDa markers; 1: ADH, 65 hoursat 6° C.; 2: ADH/cNAD, 65 hours at 6° C.; 3: ADH/NAD, 65 hours at 6° C.;4: ADH, 65 hours at 35° C.; 5: ADH/cNAD, 65 hours at 35° C.; 6: ADH/NAD,65 hours at 35° C.

FIG. 13 shows an analysis of alcohol dehydrogenase from yeast by gelelectrophoresis after storage at 35° C. in the presence of NAD and cNAD,respectively. Test conditions: MW, 10 220 kDa markers; 1: ADH/NAD, 0days; 2: ADH/NAD, 1 day; 3: ADH/NAD, 2 days; 4: ADH/NAD, 3 days; 5:ADH/NAD, 5 days; 6: ADH/cNAD, 0 days; 7: ADH/cNAD, 1 day; 8: ADH/cNAD, 2days; 9: ADH/cNAD, 3 days; 10: ADH/cNAD, 6 days.

FIG. 14 is a depiction of the stability of alcohol dehydrogenase fromyeast in the presence of NAD and cNAD, respectively, in liquid phase at35° C. over a period of 65 hours. Test conditions: ADH 5 mg/ml; NAD andcNAD, respectively, 50 mg/ml; buffer: 75 mM Na4P2O7, glycine, pH 9.0;temperature 35° C.

FIG. 15 is a depiction of various function plots of glucosedehydrogenase after storage in the presence of NAD and various mediatorsat room temperature for 11 weeks.

FIG. 16 is a depiction of the results of the glucose dehydrogenaseenzyme kinetics in the presence of NAD and1-(3-carboxypropoxy)-5-ethylphenazinium trifluoro-methanesulfonate atvarious glucose concentrations.

FIG. 17 is a diagrammatic depiction of glucose detection with GlucDH asenzyme and diaphorase as mediator.

FIG. 18 is a depiction of the function plots of glucose dyeoxidoreductase (GlucDOR) in the presence of pyrroloquinolinequinone(PQQ) and [(4-nitroso-phenyl)imino]dimethanol hydrochloride as mediator,and of glucose dehydrogenase in the presence of NAD anddiaphorase/[(4-nitrosophenyl)imino]dimethanol hydrochloride as mediator,respectively.

FIG. 19 is a depiction of the results of the glucose dehydrogenaseenzyme kinetics in the presence of NAD and diaphorase at various glucoseconcentrations.

FIG. 20 is a depiction of the current measured as a function of theglucose concentration in the electrochemical determination of glucoseusing glucose dehydrogenase in the presence of NAD and cNAD,respectively. Test conditions: 25 mM NAD and cNAD, respectively; 2.5seconds delay; 5 seconds measurement time.

FIG. 21 is a depiction of the amino acid sequences of the glucosedehydrogenase double mutants GlucDH_E96G_E170K and GlucDH_E170K_K252L.

In order that the present invention may be more readily understood,reference is made to the following detailed descriptions and examples,which are intended to illustrate the present invention, but not limitthe scope thereof.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENT INVENTION

The following descriptions of the embodiments are merely exemplary innature and are in no way intended to limit the present invention or itsapplication or uses.

Example 1

Carba-NAD (FIG. 1A) or NAD were added to the glucose-specific GlucDH.These formulations were in each case applied to Pokalon film (Lonza)and, after drying, stored under warm and moist conditions (32° C., 85%relative humidity). The reaction kinetics and the function plot weresubsequently determined at regular intervals. In parallel, at each ofthe times of measurement a cNAD/NAD analysis and a determination of theresidual activity of the enzyme were carried out.

The kinetics plots for NAD (FIG. 2A) and cNAD (FIG. 2B) determined onthe first day are comparable and also show a similar rise in the glucosedependence. However, a distinct difference in the kinetics plots isevident after 5 weeks. Whereas the kinetics for NAD (FIG. 2C) decreasegreatly in their dynamics, the kinetics of the enzyme stabilized withcNAD remain virtually unchanged (FIG. 2D).

There is also a distinct difference in the blank values (dry blank valuebefore application of a blood sample), as is evident from FIG. 3. Therise in the dry blank value for NAD is attributable to the formation offluorescent particles (Oppenheimer (1982), Supra). Surprisingly, thisdoes not occur with cNAD.

The differing stability of glucose dehydrogenase in the presence of NADand cNAD, respectively, is also evident from comparison of FIGS. 4 and5. After 5 weeks, the function plot for the enzyme stabilized with cNADis still in the bunch of previous measurements (FIG. 5A), whereas theplot for the enzyme treated with NAD (FIG. 4) shows a fall-off at higherconcentrations, which is a typical sign that the amounts ofenzyme/coenzyme are too low. FIG. 5B shows various function plots of theglucose dehydrogenase stabilized with cNAD over a period of 24 weeks. Itis clear in this connection that the function of the enzyme is onlyslightly changed at high glucose concentrations throughout the periodand approximately corresponds after 24 weeks to the value obtained after5 weeks.

The relation between structure of the coenzyme and its stability over apredetermined period is evident from FIG. 6. According to this, theresidual content of cNAD in a glucose detection reagent after storage(at 32° C. and 85% relative humidity) for 24 weeks is still about 80% ofthe initial level, whereas the content of NAD in a glucose detectionreagent stabilized with NAD declines after only 5 weeks to about 35% ofthe initial level and, by extrapolation, is reduced to zero after about17 weeks.

The result of the determination of residual activity of the activeGlucDH enzyme after 5 weeks at 32° C. and 85% relative humidity (FIG.7A) is completely surprising. The enzyme stabilized with NAD now showsonly an extremely low enzyme activity (0.5%), whereas the enzymestabilized with cNAD still has a residual activity of 70% (in each caseby comparison with samples stored in a refrigerator with desiccant).After 24 weeks at 32° C. and 85% relative humidity (FIG. 7B), theresidual activity of the enzyme on stabilization with cNAD is stillabout 25%.

If a mutant is used instead of the wild-type enzyme (from Bacillussubtilis), it is possible to increase the residual GlucDH activity evenfurther. After storage at 32° C. and 85% relative humidity in thepresence of cNAD for 24 weeks, the residual activity of aGlucDH_E96G_E170K mutant with the amino acid replacements glutamicacid→glycine at position 96 and glutamic acid→lysine at position 170(GlucDH-Mut1) of the wild-type enzyme is about 70%, whereas the residualactivity of a GlucDH_E170K_K252L mutant with the amino acid replacementsglutamic acid→lysine at position 170 and lysine→leucine at position 252(GlucDH-Mutt) is about 50% (FIG. 8).

The analysis of glucose dehydrogenase by gel electrophoresis in an SDSgel (FIGS. 9 and 10) also shows clearly the difference between storagein the presence of NAD and cNAD, respectively. Whereas the enzyme isstill identifiable as a band with the expected mobility after storagefor 5 weeks at 32° C. and 85% relative humidity in the presence of cNAD,the enzyme stored in the presence of NAD has completely disappeared(FIG. 9). It is evident at the same time from FIG. 10 that the band ofthe enzyme stabilized by NAD and stored at 50° C. becomes weaker as thestorage time increases and has virtually disappeared after 476 hours,whereas the corresponding band of the enzyme stored in the presence ofcNAD shows an only slight change compared with a band detected at thestart of the experiment.

This result can also be confirmed on storage in liquid phase (FIGS. 11Aand 11B). After 95 hours at 50° C., the residual activity of glucosedehydrogenase in the presence of the native coenzyme NAD is 5%, whereasthe residual activity of GlucDH in the presence of the artificialcoenzyme cNAD is 75% (FIG. 11A). After storage at 50° C. for 336 hours,the residual activity of the enzyme stabilized with NAD is now onlyabout 1%; the residual activity of the enzyme stored in the presence ofcNAD is observed to be still about 70%. The corresponding SDS gelslikewise show a change in the GlucDH band in the presence of the nativecoenzyme NAD: new bands appear at higher molecular masses and there is ashift in the 30 kDa band.

Overall, it is an extremely surprising result that stabilization of thecofactor simultaneously brings about a stabilization of the enzyme—andnot just through the cooperative effect of the better cohesion of theenzyme. Decomposition of the cofactor NAD has a negative effect on thestability of the enzyme GlucDH and in fact speeds up inactivationthereof. Replacement of native NAD by artificial analogues permitsGlucDH to be stored under stress conditions (e.g. elevated temperature)even in the presence of a cofactor.

It is possible with such a system to produce blood glucose test stripswith considerably improved stability properties, for which apresentation without desiccant is possible.

Example 2

cNAD or NAD were added to an alcohol detection solution. These mixtureswere stored at 35° C. The stability of the enzyme was then checked atregular intervals, and the residual activity of the enzyme wasdetermined.

Once again, an analysis by gel electrophoresis in an SDS gel (FIGS. 12and 13) shows the difference between storage in the presence of NAD andcNAD. Thus, the bands of the alcohol dehydrogenase stabilized with cNADdiffer only slightly after storage at 6° C. and 35° C., respectively,for 65 hours, indicating a stabilization of the enzyme by artificialcoenzyme. By contrast, the band of the enzyme stored in the presence ofNAD at 35° C. has completely disappeared (FIG. 12).

It is further clear from FIG. 13 that the band of the enzyme stabilizedwith NAD and stored at 35° C. becomes weaker as the storage timeincreases and has almost completely disappeared after 5 days. A band ofthe enzyme stabilized with cNAD detected after storage at 35° C. for 6days shows distinctly less decomposition of the enzyme and thus anincreased stability.

This result can be confirmed also on storage in liquid phase (FIG. 14).After 65 hours at 35° C., the residual activity of alcohol dehydrogenasein the presence of the native coenzyme NAD is about 6%, whereas theresidual activity of the enzyme in the presence of the artificialcoenzyme cNAD is still about 60%.

Example 3

To determine glucose, various test systems which included in each caseglucose dehydrogenase, NAD, a mediator and, where appropriate, anoptical indicator were measured photometrically and electrochemically.

For photometric measurements, initially four test elements which had ineach case been stored at room temperature for 11 weeks and comprised2,18-phosphomolybdic acid, besides glucose dehydrogenase, NAD and amediator, were investigated with various glucose concentrations.

As is evident from FIG. 15, a fall in the reflectance was observed withincreasing glucose concentration for all four mediators employed, i.e.[(4-nitrosophenyl)imino]dimethanol hydrochloride (Med A),1-methyl-5,6-dioxo-5,6-dihydro-1,10-phenanthroliniumtrifluoromethanesulfonate (Med B),7-methyl-5,6-dioxo-5,6-dihydro-1,7-phenanthroliniumtrifluoromethanesulfonate (Med F) and1-(3-carboxypropoxy)-5-ethylphenazinium trifluoromethanesulfonate (MedG), and thus the abovementioned mediators are in principle suitable fordetermining glucose by photometry.

At high glucose concentrations in the region of 800 mg/dl, thereflectance of the measured sample on use of[(4-nitrosophenyl)imino]dimethanol hydrochloride and1-(3-carboxypropoxy)-5-ethylphenazinium trifluoromethanesulfonate,respectively, is still about 20%, suggesting that these two mediatorsare particularly suitable for photometric measurements using the glucosedehydrogenase/NAD system, and thus also the glucose dehydrogenase/cNADsystem. The kinetics of the conversion of glucose using the glucosedehydrogenase, NAD, 1-(3-carboxypropoxy)-5-ethylphenaziniumtrifluoromethanesulfonate and 2,18-phosphomolybdic acid system atglucose concentrations in the range from 0 to 800 mg/dl are depicted inFIG. 16.

As is evident from the diagrammatic depiction in FIG. 17, thephotometric determination of glucose can also take place with(additional) use of diaphorase as intermediary mediator. FIG. 18 shows aconcentration-dependent decrease in reflectance for the glucosedehydrogenase, NAD, diaphorase, [(4-nitrosophenyl)imino]dimethanolhydrochloride and 2,18-phosphomolybdic acid system (system 1). Thesystem which served as comparison was glucose dye oxidoreductase,pyrroloquinolinequinone, [(4-nitrosophenyl)imino]dimethanolhydrochloride and 2,18-phosphomolybdic acid (system 2), which likewisecauses a concentration-dependent decrease in the reflectance, but hasdisadvantages because of the low specificity of glucose dyeoxidoreductase. The kinetics of the conversion of glucose using system 1at glucose concentrations in the range from 0 to 800 mg/dl are depictedin FIG. 19.

As alternative to photometry it is also possible to employ anelectrochemical measurement for the purpose of determining analytes.Thus, the current required to reoxidize the reduced mediator was foundto be linearly dependent on the glucose concentration (FIG. 20) bothwith a test element which, besides glucose dehydrogenase, comprised NADas coenzyme and 1-(3-carboxypropoxy)-5-ethylphenaziniumtrifluoromethanesulfonate as mediator, and with a corresponding systemwhich included the stabilized coenzyme cNAD instead of NAD.

It has thus been shown that determination of analytes using thedehydrogenase/stable coenzyme system is also possible by means ofelectrochemical detection and evaluation with another wavelength whichis independent of the coenzyme. The overall formulation ought also to befurther stabilized through the use of the stable enzyme/coenzyme pair.

The features disclosed in the above description, the claims and thedrawings may be important both individually and in any combination withone another for implementing the invention in its various embodiments.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation may vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Having described the present invention in detail and by reference tospecific embodiments thereof, it will be apparent that modification andvariations are possible without departing from the scope of the presentinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of thepresent invention.

1. A method for stabilizing an enzyme, wherein the enzyme is stored inthe presence of a stable coenzyme.
 2. The method according to claim 1,wherein the enzyme is selected from dehydrogenases.
 3. The methodaccording to claim 2, wherein the enzyme comprises a dehydrogenaseselected from the group consisting of glucose dehydrogenase(E.C.1.1.1.47), lactate dehydrogenase (E.C.1.1.1.27, 1.1.1.28), malatedehydrogenase (E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6),alcohol dehydrogenase (E.C.1.1.1.1), alpha-hydroxybutyratedehydrogenase, sorbitol dehydrogenase, and L-amino-acid dehydrogenase(E.C.1.4.1.5).
 4. The method according to claim 3, wherein the enzymecomprises glucose dehydrogenase.
 5. The method according to claim 1,wherein the stable coenzyme is selected from stable nicotinamide adeninedinucleotide (NAD/NADH) compounds and nicotinamide adenine dinucleotidephosphate (NADP/NADPH) compounds and the compound of the formula (I)


6. The method according to claim 5, wherein the stable coenzyme isselected from compounds having the general formula (II)

with A=adenine or an analogue thereof, T=in each case independently O,S, U=in each case independently OH, SH, BH₃ ⁻, BCNH₂ ⁻, V=in each caseindependently OH or a phosphate group, or two groups forming a cyclicphosphate group; W═COOR, CON(R)₂, COR, CSN(R)₂ with R=in each caseindependently H or C₁-C₂-alkyl, X¹, X²=in each case independently O,CH₂, CHCH₃, C(CH₃)₂, NH, NCH₃, Y═NH, S, O, CH₂, Z=a linear or cyclicorganic radical, with the proviso that Z and the pyridine residue arenot linked by a glycosidic linkage, or a salt or, where appropriate, areduced form thereof.
 7. The method according to claim 6, in which Z isselected from (i) a linear radical having 4-6 C atoms, in which 1 or 2atoms are optionally replaced by one or more heteroatoms selected fromO, S and N, and (ii) a radical including a cyclic group having 5 or 6 Catoms, which optionally comprises a heteroatom selected from O, S and Nand optionally one or more substituents, and a radical CR⁴ ₂, where CR⁴₂ is bonded to the cyclic group and to X², with R⁴=in each caseindependently H, F, Cl, CH₃.
 8. The method according to claim 7, inwhich Z is a compound of the general formula (III)

where a single or double bond may be present between R^(5′) and R^(5″),with R⁴=in each case independently H, F, Cl, CH₃, R⁵═CR⁴ ₂, whereR^(5′)═O, S, NH, NC₁-C₂-alkyl, CR⁴ ₂, CHOH, CHOCH₃, and R^(5″)═CR⁴ ₂,CHOH, CHOCH₃ if there is a single bond between R^(5′) and R^(5″), whereR^(5′)═R^(5″)═CR⁴ if there is a double bond between R^(5′) and R^(5″),and R⁶, R^(6′)=in each case independently CH or CCH₃.
 9. The methodaccording to claim 6, in which W═CONH₂ or COCH₃.
 10. The methodaccording to claim 8, in which R⁵ is CH₂.
 11. The method according toclaim 8, in which R^(5′) is selected from CH₂, CHOH and NH.
 12. Themethod according to claim 8, in which R^(5′) and R^(5″) are each CHOH.13. The method according to claim 8, in which R^(5′) is NH and R^(5″) isCH₂.
 14. The method according to claim 1, wherein the stable coenzyme iscarbaNAD.
 15. The method according to claim 1, wherein the enzymestabilized with a stable coenzyme is stored for a period of at least 2weeks.
 16. The method according to claim 1, wherein the enzymestabilized with a stable coenzyme is stored at a temperature of at least20° C.
 17. The method according to claim 1, wherein the enzymestabilized with a stable coenzyme is stored without a drying reagent.18. The method according to claim 1, wherein the enzyme stabilized witha stable coenzyme is stored at a relative humidity of at least 50%. 19.The method according to claim 1, wherein the storage of the enzymestabilized with a stable coenzyme takes place as dry substance.
 20. Themethod according to claim 1, wherein the storage of the enzymestabilized with a stable coenzyme takes place in liquid phase.
 21. Themethod according to claim 1, wherein the storage of the enzymestabilized with a stable coenzyme takes place on a test element.
 22. Anenzyme stabilized with a stable coenzyme, wherein said enzyme shows adecline in the enzymatic activity of less than 50% compared with theinitial level upon storage for at least 2 weeks at a temperature of atleast 20° C. with high humidity and without a drying reagent, whereinthe enzyme is a mutated glucose dehydrogenase and the mutated glucosedehydrogenase has an increased thermal or hydrolytic stability comparedwith the corresponding wild-type glucose dehydrogenase.
 23. A detectionreagent for determining an analyte, which detection reagent comprises astabilized enzyme according to claim
 22. 24. A test element comprising adetection reagent for determining an analyte, which detection reagentcomprises a stabilized enzyme according to claim 22.